Selective separation or extraction of steroidal glycosides by supercritical fluid extraction using carbon dioxide

ABSTRACT

A method for selectively separating desirable steroidal glycosides from undesirable components present in plant material of the Asclepiadaceae family containing the same. The method comprises contacting the plant material or material derived therefrom (herein: “the material”) with liquid or supercritical carbon dioxide under conditions whereby the desirable steroidal glycosides dissolve in the liquid or supercritical carbon dioxide in preference to the undesirable components, and subsequently recovering the desirable steroidal glycosides from the carbon dioxide solution.

FIELD OF THE INVENTION

The present invention relates to the selective separation or extractionof steroidal glycosides from materials containing more than one of thesecompounds and optionally other components. Most particularly, suchmaterials may be plant matter in which the steroidal glycosides occurnaturally.

BACKGROUND OF THE INVENTION

Extracts obtainable from plants of the Asclepiadaceae family,particularly the Hoodia genus (formerly the Hoodia and Trichocaulongenera), have been shown to contain the steroid glycoside 1 which is anappetite suppressing compound (PCT application WO 98/46243). Thecontents of this prior application are incorporated herein by reference.An extraction process is outlined in the prior application, involvingtreating plant material with a solvent to extract a fraction havingappetite suppressant activity, separating the extraction solution fromthe rest of the plant material, removing the solvent from the extractionsolution and recovering the extract. The solvents specifically mentionedto perform the extraction are one or more of methylene chloride(dichloromethane), water, methanol, hexane, ethyl acetate or mixturesthereof.

The prior art solvent extraction process tends, however, to result in amixture of extracted compounds, not all of which have desirableproperties. Typically, some may have an unpleasant taste.

The need for a subsequent fractionation or separation procedure toremove undesirable extracted compounds leads to increased manufacturingcosts and complicated procedures, which it is preferable to avoid.

US Patent Application No. US-A-2003/0152648, the disclosure of which isincorporated herein by reference, describes extraction of pregnaneglycosides, particularly stavarosides, from the plant Orbea variegata, amember of the Asclepiadaceae family. The extraction process involvesinitial solvent extraction from dried and ground plant material, using asuitable solvent. It is stated generally that “suitable solvents includewater, dilute acids, organic solvents, critical, supercritical or nearcritical fluid solvents, e.g. carbon dioxide, nitrous oxide, propane,ethane, ethylene and fluorohydrocarbons, and mixtures of any of these”.The preferred solvent is stated to be an alcohol based solvent, the mostpreferred being ethanol. Ethanol extraction is the only technique shownin any working example. The solvent extraction technique is stated to beusable to extract stavarosides from the plant material, and there is noteaching or suggestion to use it to selectively separate desirablesteroidal glycosides from undesirable steroidal glycosides.

Moore and Taylor (J. Nat. Prod 1996, 59, 690-693), the disclosure ofwhich is incorporated herein by reference, describe the extraction ofthe cardiac glycosides, digoxin and acetyldigoxin, from Digitalis lanatausing near supercritical methanol-modified carbon dioxide. The authorsindicate that to effect efficient extraction the extraction solvent wasmethanol and carbon dioxide where the methanol was in the range of10-25%, with the optimised conditions employing 20% methanol modifiedCO₂

We have now found, surprisingly, that, by using a particular extractiontechnique described below, specifically the use of liquid orsupercritical carbon dioxide, a relatively efficient selectiveextraction of desirable steroidal glycosides from Asclepiadaceae plantmaterial can be achieved, which involves the selective separation ofdesirable steroidal glycosides from undesirable steroidal glycosides,reducing or eliminating the need for subsequent processing to removeundesirable components.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides in the first aspect a method forselectively separating desirable steroidal glycosides from undesirablesteroidal glycosides or other compounds present in plant material of theAsclepiadaceae family containing the same, the method comprisingcontacting the plant material or material derived therefrom (herein:“the material”) with liquid or supercritical carbon dioxide underconditions whereby the desirable steroidal glycosides dissolve in theliquid or supercritical carbon dioxide in preference to the undesirablesteroidal glycosides or other compounds, and subsequently recovering thedesirable steroidal glycosides from the carbon dioxide solution.

The use of liquid or supercritical carbon dioxide as an extractionsolvent is known, and apparatus for performing the technique are readilyavailable. For a general review of the technology, please refer toKaiser et al, Pharmazie 56, 907-926 (2001) and Lang et al, Talanta 52,771-782 (2001), the contents of which are incorporated herein byreference. The conditions of the extraction which may be varied tocontrol the selectivity are primarily the temperature and the pressureof the sealed extraction apparatus. However, the prior art has generallyrestricted the use of the technique to extraction of active compoundsfrom inactive cell-wall and other material and the use of liquid orsupercritical carbon dioxide as a means for selectively separatingdesirable steroidal glycosides from relatively chemically similarundesirable steroidal glycosides in plant material of the Asclepiadaceaefamily was not previously known or suggested.

After an initial extraction from the material, further extraction cyclescan be performed on the remaining material if desired, using freshcarbon dioxide, in order to maximise the yield of extracted compounds.

An initial extraction can be performed using liquid carbon dioxide, anda subsequent extraction performed on the remaining material usingsupercritical carbon dioxide. Alternatively, both an initial and asubsequent extraction can use liquid carbon dioxide. Still further, bothan initial and a subsequent extraction can use supercritical carbondioxide. The conditions of these sequential extractions can be chosen topreferentially remove particular components from the material.

The method of the present invention may, if desired, be performed usinga flow of carbon dioxide with downstream recovery of the desirablesteroidal glycosides.

The method of the present invention can be used with other extractionprocedures for removing other, e.g. non-steroidal, components from thematerial to be treated. Thus, for example, an initial extraction usingliquid carbon dioxide can be performed under conditions selected topreferentially remove undesirable lipophilic compounds such as fattyacids from the material. A subsequent extraction performed on theremaining material according to the present invention can thereby resultin an improved yield of the desirable steroidal glycoside.

An extract produced by the method of the invention can, if desired, besubjected to one or more subsequent extractions, e.g. one or moresubsequent solvent extractions using one or more organic solvent toremove any residual unwanted compounds, for example non-steroidalglycosides. The choice of organic solvent(s) will be well within thecapacity of a person of ordinary skill in this art, considering thematerials being treated. The said one or more subsequent solventextractions may suitably be performed using organic solvents selectedfrom acetone, ethyl acetate, heptane (e.g. n-heptane), ethanol, hexane(e.g. n-hexane) and any mixture or combination thereof, and at anyconvenient temperature.

The material on which the method of the present invention may beperformed may be plant material or material derived therefrom. The term“plant material” in this context includes all forms of plant material,including freshly cut plant material, dried or preserved plant material,which may optionally be comminuted, e.g. powdered or crushed. The term“material derived therefrom” in this context includes extracts anddecoctions obtained from the plant material. Such extracts may, forexample, be obtainable by a non-selective extraction method, such as theprior art solvent extraction method.

In one embodiment, the desirable steroidal glycoside may comprise asteroidal glycoside having a desirable bioactivity, for example havingbioactivity to suppress appetite (particularly through actions atcentres in the brain controlling of feelings of hunger and fullness), totreat excessive body weight (particularly above-average body weight foran individual's age and height), to treat obesity, to reduce totalcalorific intake of an individual (particularly, total calorific intakeover an extended period of at least about two weeks in a lifestyle orenvironment where calorific foodstuffs and beverages are availablesubstantially ad libitum), or any combination thereof. Moreparticularly, the steroidal glycoside may be a compound of formula 1 asset out above, and the plant may be a plant of the Hoodia genus. Theplant of the Hoodia genus may suitably be selected from Hoodia gordonii,Hoodia currorii subsp. currorrii and Hoodia currorii subsp. lugardii.The plant of the Hoodia genus is preferably Hoodia gordonii.

The undesirable steroidal glycosides or other compounds, from which thedesirable steroidal glycoside is to be separated according to the methodof the present invention, may, for example, comprise an unpleasanttasting component.

A multi-step extraction process may use conditions of differingtemperature and/or pressure at different stages. Most particularly,conditions of differing pressure may be used. The present inventiontherefore includes within its scope the use of a multi-pressureextraction of dried plants of the Hoodia genus wherein the plantmaterial is initially extracted with carbon dioxide under liquidconditions to extract unwanted lipophilic material such as fatty acids,and then subsequently extracted with carbon dioxide under supercriticalconditions to afford an extract containing elevated levels of compound1, whilst leaving unwanted steroidal glycosides or other compounds withthe plant matrix.

If desired, one or more aqueous or organic co-solvent may be used in themethod of the present invention, in conjunction with the carbon dioxidesolvent and under the same conditions of temperature and pressure asapplied to the carbon dioxide. Any such co-solvent that is present willbe used in generally small amounts relative to the carbon dioxide, forexample less than about 10% by weight (e.g. about 5% by weight) relativeto the amount of carbon dioxide used. The co-solvent may suitably beselected from water, methanol, ethanol, hexane (e.g. n-hexane), heptane(e.g. n-heptane) and any mixture or combination thereof.

The present invention includes within its scope steroidal glycosideswhich have been separated from the material by means of the method ofthe present invention.

DETAILED DESCRIPTION OF THE INVENTION

The taxonomy of plants of the Asclepiadaceae family has been revised inrecent years. For example, in 1992 Bruyns reclassified a number ofplants to the genus Hoodia, which were previously in the genusTrichocaulon (Bruyns; Bot. Jahrb. Syst. 115 (2) 145-270 (1993)). Onesuch reclassification related to Hoodia pilifera, previouslyTrichocaulon piliferum. The genus Trichocaulon now does not exist.

Work undertaken on identifying molecules present in plants belonging tothe genus Hoodia has shown that compound 1 is present in addition to aset of steroid glycosides covered by the generic structure 2.

For example, from Hoodia gordonii eleven compounds have been isolatedand characterised (Compounds 3-13).

Compound R₁ R₂ 3 glu-glu-glu- tig-the-ole- 4 glu-glu- tig-mda-cym- 5glu-glu-glu- tig-cym-cym 6 glu-glu-glu- tig-ole-cym-cym- cym- 7glu-glu-glu- tig-ole-cym- 8 glu-glu-glu- ang-mda-cym- 9 glu-glu-glu-tig-the-cym- 10 glu-glu-glu- tig- 11 glu-glu-glu- tig-cym- 12glu-glu-glu- tig-the-ole-cym- 13 glu-glu-glu- tig-the-cym-cym- ole =oleandrose, cym = cymarose, glu = glucose, mda =3-O-methyl-6-deoxyallose, tig = tigloyl, ang = angeloyl, the =thevetose.

Methanol extraction of dried Hoodia gordonii plant material affordsextracts containing compound 1 and compounds 3-13. The use of othersolvents such as dichloromethane or ethyl acetate similarly producesextracts containing all the steroidal glycosides.

Surprisingly, it has been found that liquid or supercritical carbondioxide extraction (for example at pressures of about 300 to about 500bar and at a temperature of about 55 to about 80° C.) affords selectiveextraction of compound 1 whilst retaining compounds 3-13 in the plantmatrix. The pressure and temperature can be varied outside these limits.

Certain members of the genus Hoodia are known to contain bittercomponents making the palatability of the plant or extracts thereofpoor. Indeed, the native names for Hoodia gordonii and Hoodia curroriisubsp. lugardii is ‘bitterghaap’ which may be translated as bitter food.The use of Hoodia gordonii as an appetite suppressing agent could behindered by this bitterness. Surprisingly, it has been found thatextraction of plants of the genus Hoodia using carbon dioxide extractionaffords an extract rich in compound 1 but lacking any unpleasant taste.

A second aspect of the invention is the use of a multi-pressureextraction of dried plants of the Hoodia genus wherein the plantmaterial is initially extracted with carbon dioxide under liquidconditions to extract unwanted lipophilic material such as fatty acids,and then subsequently extracted with carbon dioxide under supercriticalconditions to afford an extract containing elevated levels of compound1, whilst leaving unwanted steroid glycosides, for example compounds3-13 with the plant matrix.

BRIEF DESCRIPTION OF THE DRAWING

The accompanying drawing shows, purely by way of illustration andwithout limitation, a suitable carbon dioxide extraction system for usein performing the present invention.

DETAILED DESCRIPTION OF THE DRAWING

A suitable extraction unit (see drawing) consists of extractor andseparator vessels as well as of different heat exchangers, pumps,regulation valves and devices. The extractor vessel is filled with theraw material which is to be treated. The required pressure is achievedthrough a solvent which flows in from a tank. For increasing thepressure, a pump is necessary, which also takes over the transport ofthe solvent after the required extraction pressure has been achieved.The pressure is maintained by an overflow valve which opens when therequired pressure is exceeded and transports the enriched solvent to theseparator step. Usually, this separation step is connected with thestorage tank of the solvent. The pressure in the separator step and inthe storage tank will consequently be the same (with the exception offlow losses) and will correspond to the saturation (evaporation)pressure of the solvent at the respective temperature.

EXAMPLES

The following Examples illustrate, without limitation, the selectiveextraction of steroidal glycosides from plants of the Asclepiadaceaefamily using carbon dioxide as the solvent.

Example 1

Dried Hoodia gordonii plant material was milled to a fine powder (<1mm). 700 g of milled material was packed into a single extractor columnand extracted with CO₂ at 300 bar and 55° C. Table 1 indicates thequantity of extract obtained during the extraction, as well as theresults of the HPLC analysis of the extracts.

TABLE 1 Yield and composition during the extraction of dried Hoodiagordonii plant material with carbon dioxide at 300 bar and 55° C.Extraction Content of Time compound (hours) Yield (g) Total Yield (g)Total Yield % 1 (%) 0.5 18.53 18.53 2.65 0.6 1.0 7.70 26.23 3.75 1.3 2.06.88 33.11 4.73 1.1 3.0 5.28 38.39 5.48 1.4 4.0 1.21 39.60 5.66 3.0 5.02.26 41.86 5.98 2.3 6.0 0.53 42.39 6.06 Not determined

Example 2

Dried Hoodia gordonii plant material was milled to a fine powder.Particle size analysis of this material showed that 85% passed a 600 μmmesh. 700 g of milled material was packed into a single extractor columnand extracted sequentially with CO₂ at increasing pressure andtemperature. Table 2 indicates the quantity of extract obtained at thefour experimental conditions, as well as the results of the HPLC and GCanalysis of the extracts.

TABLE 2 Yield and composition under increasing pressure and temperatureconditions of carbon dioxide % of nine identified % Extracted steroid ofcom- % tri- % hydro- weight (g) glycosides pound 1 glycerides carbons 60 bar 10° C. 5.000 10.9 1.2 39.3 1.4 100 bar 13.510 43 0.7 17.2 1.325° C.-35° C. 200 bar 35° C. 1.600 36.5 8.8 11.0 14.8 300 bar 55° C.13.290 19.2 4.6 17.9 7.9

Example 3

A series of extractions were performed using standardised conditions ofextraction of milled Hoodia gordonii plant material with carbon dioxideat 100 bar at 25° C.-35° C. (LCO₂) for 7 hours, followed by extractionat 300 bar at 55° C. (SCO₂) for 7 hours. The two extracts obtained wereanalysed by HPLC to determine that levels of steroid glycosides. Table 3shows the quantity of input material per batch and the percentage ofcompound 1 in the two extracts obtained for each of the extractionexperiments.

The input batches of dried Hoodia gordonii contain 0.06-0.2% ofcompound 1. Extraction with liquid carbon dioxide (LCO2) affords anextract in a yield of 0.71-1.94% that contains 0.6-2.7% of compound 1.Compounds 3-13 were not detected in the product. Sequential extractionwith supercritical carbon dioxide (SCO2) affords a yellow-green powderwhich has no bitter taste in a yield of 0.67-2.34% that contains2.2-5.7% of compound 1. Again compounds 3-13 were not detected in theproduct.

TABLE 3 The yield and content of compound 1 obtained in the extractionof dried Hoodia gordonii plant material with sequential liquid andsupercritical carbon dioxide Experiment number 47/091 47/093 47/09447/095 47/096 47/098 47/099 Plant Input weight 693 530.2 731.5 743.3710.1 731.4 742.3 material (g) % compound 0.20 0.20 0.20 0.14 0.14 0.200.20 1 in input LCO2 Mass 8.71 4.76 7.67 9.18 6.13 6.32 9.23 obtained(g) % yield 1.26 0.90 1.05 1.24 0.86 0.86 1.24 % compound 2.7 2.0 1.41.0 1.9 1.4 1.3 1 in LCO2 SCO2 Mass 8.00 7.60 17.13 14.20 7.20 10.7010.30 obtained (g) % yield 1.15 1.43 2.34 1.91 1.01 1.46 1.39 % compound5.4 5.0 5.2 4.6 4.5 5.7 5.6 1 in SCO2 Experiment number 47/100 47/10147/102 47/103 47/104 47/110 Plant Input weight 736.3 755.3 733.1 790.4785 730 material (g) % compound 0.16 0.16 0.20 0.20 0.06 0.19 1 in inputLCO2 Mass 7.98 7.48 8.2 6.02 5.61 14.15 obtained (g) % yield 1.08 0.991.12 0.76 0.71 1.94 % compound 0.8 1.4 1.1 1.7 0.6 0.7 1 in LCO2 SCO2Mass 11.00 10.60 14.14 11.48 5.27 9.80 obtained (g) % yield 1.49 1.401.93 1.45 0.67 1.34 % compound 4.7 4.6 4.2 5.2 2.2 3.2 1 in SCO2

Example 4

A series of extractions were performed using standardised conditions ofextraction of milled Hoodia gordonii plant material with carbon dioxideat a pressure of 100 bar. Material was milled using a coffee grinder andsieved to between 106 and 600 μm. The ground material was dried at 70°C. for a minimum of 16 hours to remove any moisture. The series ofexperiments used plant material of 650-810 g. The extraction pressurewas maintained at 100 bar with a CO₂ flow rate of 5 kg/h for anextraction period of 7 hours. The product was removed from the separatorby washing with methanol. The methanol washings were evaporated todryness to obtain an accurate value for the extraction mass yield. Theextraction efficiency of compound 1 was obtained by HPLC analysis of themethanol washings and compared to the amount of compound 1 in the inputmaterial. Table 4 shows the quantity of input material per batch and thepercentage of compound 1 in the extracts obtained for each of theextraction experiments where the extraction temperature was varied from5 to 75° C. Compounds 3-13 were not detected in the product.

TABLE 4 The extraction efficiency for compound 1 obtained in theextraction of dried Hoodia gordonii plant material at a variety oftemperatures with liquid or supercritical carbon dioxide at a fixedpressure of 100 bar Charge Mass of Compound Extraction weightTemperature extract 1 in efficiency (g) (° C.) (g) extract (%) forcompound 1 (%) 807.6 5 9.69 2.75 18.33 717.4 15 6.60 2.71 13.85 707.4 258.77 1.53 10.54 712.5 35 8.69 0.46 3.12 725.7 40 11.97 0.15 1.65 723.745 3.62 0 0.00 657.6 55 0.59 0.14 0.07

Example 5

A series of extractions were performed on milled Hoodia gordonii plantmaterial with carbon dioxide at a pressure of 300 bar. Material wasmilled using a hammermill passing through a 2 mm mesh sieve. Theextraction pressure was maintained at 300 bar with a CO₂ feed ratio of100 kg CO_(2/)kg input for an extraction period of ca 7 hours. Theextraction efficiency of compound 1 was obtained by HPLC analysis of thespent plant material and compared to the amount of compound 1 in theinput material. Table 5 shows the quantity of input material per batchand the extraction efficiency of compound 1 for each of the extractionexperiments where the extraction temperature was varied from 0 to 90° C.Compounds 3-13 were not detected in the product.

TABLE 5 The extraction efficiency for compound 1 obtained in theextraction of dried Hoodia gordonii plant material at a variety oftemperatures with liquid or supercritical carbon dioxide at a fixedpressure of 300 bar Charge Temperature Extraction efficiency weight (g)(° C.) for compound 1 (%) 87.54 0 27 88.55 20 31 87.52 40 48 85.92 60 5385.02 80 59 84.75 90 60

Example 6

A series of extractions were performed on milled Hoodia gordonii plantmaterial with supercritical carbon dioxide exploring the effects of hightemperatures and pressures, the input particle size, and the presence ofco-solvents. Material was milled using a hammermill passing througheither a 0.7, 1 or 2 mm mesh sieve. The extraction pressure wasmaintained set at either 300 or 500 bar with a CO₂ feed ratio of 100 kgCO₂/kg input for an extraction period of ca 7 hours. The extractionefficiency of compound 1 was obtained by HPLC analysis of the spentplant material and compared to the amount of compound 1 in the inputmaterial. Table 6 shows the quantity of input material per batch and theextraction efficiency of compound 1 for each of the extractionexperiments. Compounds 3-13 were not detected in the product.

TABLE 6 The extraction efficiency for compound 1 obtained in theextraction of dried Hoodia gordonii plant material at a variety oftemperatures, pressures, input particle size with supercritical carbondioxide in the presence or absence of co-solvents Extraction Pre-efficiency treatment Presence for Charge sieve size Temperature Pressureof compound 1 weight (g) (mm) (° C.) (bar) co-solvent (%) 85.02 2 80 300None 59 84.92 1 80 300 None 63 84.41 0.7 80 300 None 70 42.08 2 80 300None 62 85.76 1 80 300 5% EtOH 80 84.29 1 80 500 None 81 83.23 0.7 80500 None 83 82.88 0.7 80 500 5% EtOH 88 83.86 0.7 80 500 None 78 84.080.7 80 500 5% EtOH 84 77.14 0.7 80 500 None 84 61.76 0.7 80 500 5% EtOH84 59.34 0.7 100 500 None 84 78.33 0.7 80 500 5% EtOH 86 65.45 0.7 80500 5% EtON 80

The foregoing broadly describes the present invention, withoutlimitation. Variations and modifications as will be readily apparent tothose of ordinary skill in the art are intended to be included in thescope of this application and subsequent patent(s).

1. A method for selectively separating desirable steroidal glycosidesfrom undesirable steroidal glycosides or other compounds present inplant material of the Asclepiadaceae family containing the same, themethod comprising contacting the plant material or material derivedtherefrom (herein: “the material”) with liquid or supercritical carbondioxide under conditions whereby the desirable steroidal glycosidesdissolve in the liquid or supercritical carbon dioxide in preference tothe undesirable steroidal glycosides or other compounds, andsubsequently recovering the desirable steroidal glycosides from thecarbon dioxide solution.
 2. A method according to claim 1, whereinpredominantly fatty acids and other lipid material are extracted in afirst extraction using liquid carbon dioxide at a first pressure,followed by extraction of steroidal glycosides using supercriticalcarbon dioxide at a second pressure whilst leaving unwanted steroidglycosides or other compounds with the plant matrix.
 3. A methodaccording to claim 1, wherein the carbon dioxide is used in conjunctionwith a relatively small amount of one or more aqueous or organiccosolvent.
 4. A method according to claim 3, wherein the co-solvent isused in an amount less than about 10% by weight of the carbon dioxide.5. A method according to claim 3, wherein the co-solvent is selectedfrom water, methanol, ethanol, hexane, heptane and any mixture orcombination thereof.
 6. A method according to claim 1, wherein thedesirable steroidal glycosides are to be used in the management of bodyweight or in the dietary control of obesity.
 7. A method according toclaim 1, wherein the desirable steroidal glycoside is extractedsubstantially free of any unpleasant tasting component of the material.8. A method according to claim 1, wherein the desirable steroidalglycoside is a compound of formula 1 as defined herein.
 9. A methodaccording to claim 8, wherein the compound of formula 1 is extractedsubstantially free of one or more steroidal glycosides selected fromcompounds formula 3 to 13 as defined herein.
 10. A method according toclaim 1, wherein the plant is a plant of the Hoodia genus.
 11. A methodaccording to claim 10, wherein the plant is selected from Hoodiagordonii, Hoodia currorii subsp. Currorrii and Hoodia currorii subsp.Lugardii.
 12. A method according to claim 11, wherein the plant isHoodia gordonii.
 13. A method according to claim 10, wherein theundesirable bitter tasting components present in plants of the genusHoodia are relatively not extracted whereas the steroidal glycosides tobe used for the management of body weight or in the dietary control ofobesity are relatively extracted.
 14. A method according to claim 1,wherein an extract initially produced is subjected to one or moresubsequent solvent extractions using one or more organic solvent toremove any residual unwanted compounds.
 15. A method according to claim14, wherein the one or more organic solvent is selected from acetone,ethyl acetate, heptane, ethanol, hexane and any mixture or combinationthereof.
 16. A method according to claim 14, wherein the one or moreorganic solvent is selected from acetone, heptane and any mixtures orcombination thereof.
 17. A steroidal glycoside obtained by a methodaccording to claim 1.